The interaction between IgE-Fc (Fc⑀) and its high affinity receptor Fc⑀RI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fc⑀-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fc⑀ and IgG1-Fc (Fc␥). Fc⑀ was found to be significantly less stable than Fc␥ under all pH and NaCl conditions tested. Additionally, the C⑀3C⑀4 domains of Fc⑀ were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fc⑀ and an Fc␥-Fc⑀RI␣ fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fc⑀ for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (K D < 10 ؊12 M). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fc⑀ unfolded, and interaction of Fc⑀ with the Fc␥-Fc⑀RI␣ fusion protein was abrogated. The unusual pH sensitivity of Fc⑀ may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.
The photoreceptor outer segment (OS) is the phototransductive organelle in the vertebrate retina. OS tips are regularly ingested and degraded by the adjacent retinal pigment epithelium (RPE), offsetting the addition of new disk membrane at the base of the OS. This catabolic role of the RPE is essential for photoreceptor health, with defects in ingestion or degradation underlying different forms of retinal degeneration and blindness. Although proteins required for OS tip ingestion have been identified, spatiotemporal analysis of the ingestion process in live RPE cells is lacking; hence the literature reflects no common understanding of the cellular mechanisms that effect ingestion. We imaged live RPE cells from mice (both sexes) to elucidate the ingestion events in real time. Our imaging revealed roles for f-actin dynamics and specific dynamic localizations of two BAR proteins, FBP17 and AMPH1-BAR, in shaping the RPE apical membrane as it surrounds the OS tip. Completion of ingestion was observed to occur by scission of the OS tip from the remainder of the OS, with a transient concentration of f-actin forming around the site of imminent scission. Actin dynamics were also required for regulating the size of the ingested OS tip, and the time-course of the overall ingestion process. The size of the ingested tip is consistent with the term, phagocytosis. However, phagocytosis usually refers to engulfment of an entire particle or cell, whereas our observations of OS tip scission indicate a process that is more specifically described as trogocytosis, in which one cell “nibbles” another cell.Significance Statement:The ingestion of the photoreceptor outer segment (OS) tips by the retinal pigment epithelium (RPE) is a dynamic cellular process that has fascinated scientists for 70 years. Yet its molecular mechanisms had not been addressed in living cells. We developed a live-cell imaging approach to investigate OS tip ingestion, and focused on the dynamic participation of actin filaments and membrane-shaping BAR proteins. We observed scission of OS tips for the first time, and were able to monitor local changes in protein concentration preceding, during, and following scission. Our approach revealed that actin filaments were concentrated at the site of OS scission, and were required for regulating the size of the ingested OS tip and the time-course of the ingestion process.
Addressing issues with the reproducibility of results is critical for scientific progress, but conflicting ideas about the sources of and solutions to irreproducibility are a barrier to change. Prior work has attempted to address this problem by creating analytical definitions of reproducibility. We take a novel empirical, mixed methods approach to understanding variation in reproducibility conversations, which yields a map of the discursive dimensions of these conversations. This analysis demonstrates that concerns about the incentive structure of science, the transparency of methods and data, and the need to reform academic publishing form the core of reproducibility discussions. We also identify three clusters of discussion that are distinct from the main group: one focused on reagents, another on statistical methods, and a final cluster focused the heterogeneity of the natural world. Although there are discursive differences between scientific and popular articles, there are no strong differences in how scientists and journalists write about the reproducibility crisis. Our findings show that conversations about reproducibility have a clear underlying structure, despite the broad scope and scale of the crisis. Our map demonstrates the value of using qualitative methods to identify the bounds and features of reproducibility discourse, and identifies distinct vocabularies and constituencies that reformers should engage with to promote change.
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